System and method for buoyant particle processing

ABSTRACT

A system for buoyant particle processing includes: a reaction vessel, a stirring mechanism, a set of one or more pumps, and a filter. The system can additionally or alternatively include a set of pathways and/or any other suitable component(s). A method for buoyant particle processing includes: stirring the contents of a reaction vessel; washing a set of buoyant particles; and filtering the contents of the reaction vessel. Additionally or alternatively, the method can include any or all of: preprocessing the set of buoyant particles; adding a set of inputs to the reaction vessel; washing the set of buoyant particles; repeating one or more; and/or any other suitable process(es).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/695,517, filed 9 Jul. 2018, which is incorporated herein in itsentirety by this reference. This application is related to U.S.application Ser. No. 16/004,874, filed 11 Jun. 2018, which is acontinuation-in-part of U.S. application Ser. No. 14/969,446, filed 15Dec. 2015, which claims the benefit of U.S. Provisional Application Ser.No. 62/092,019, filed on 15 Dec. 2014 and U.S. Provisional ApplicationSer. No. 62/189,518 filed on 7 Jul. 2015, which are each incorporatedherein in their entirety by this reference.

TECHNICAL FIELD

This invention relates generally to separation methods and systems inthe field of biological and chemical sample processing. Morespecifically, it relates to an improved system and method for themanufacture of buoyant particles used in the field of biological andchemical sample processing.

BACKGROUND

In research and diagnostic applications, it is often important to beable to isolate one or more types of particles of a sample. Isolation oftarget components in an efficient and high throughput manner can thushave a significant impact in healthcare applications, biologicalresearch, research in the food industry, bioprocessing, fermentation,and medical research. Components for isolation and extraction caninclude cells, proteins, nucleic acids, lipids, chemical compounds, andother particles commonly found in biological fluid. Buoyant particleshave been shown to be useful in these applications. Manufacturing andpreparing buoyant particles suitable for these applications, however,can be a very challenging process, as well as require large amounts ofuser intervention to perform.

Thus, there is a need in the biological and chemical sample processingfield to create an improved method and system for buoyant particleprocessing. This invention provides such an improved method and system.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a variation of the system 100 for buoyant particleprocessing;

FIG. 2 depicts a variation of the method 200 for buoyant particleprocessing;

FIG. 3 depicts a variation of a processed buoyant particle;

FIG. 4 depicts a specific example of a variation of a processed buoyantparticle;

FIGS. 5A-5D depict a variation of a buoyant particle at various stagesof the method 200;

FIGS. 6A-6C depict a variation of a buoyant particle at various stagesof the method 200;

FIG. 7 depicts a cross-sectional view of a variation of a tangentialflow filter;

FIG. 8 depicts a cross-sectional view of a variation of a direct flowfilter; and

FIG. 9 depicts a variation of the system 100 for buoyant particleprocessing.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description of preferred embodiments and examples of theinvention is not intended to limit the invention to these preferredembodiments and examples, but rather to enable any person skilled in theart to make and use this invention.

1. Overview

As shown in FIG. 1, a system 100 for buoyant particle processingincludes: a reaction vessel 110, a stirring mechanism 120, a set of oneor more pumps 130, and a filter 140. The system 100 can additionally oralternatively include a set of pathways 150 and/or any other suitablecomponent(s).

As shown in FIG. 2, a method 200 for buoyant particle processingincludes: stirring the contents of a reaction vessel S230; washing a setof buoyant particles S240; and filtering the contents of the reactionvessel S250. Additionally or alternatively, the method 200 can includeany or all of: preprocessing the set of buoyant particles S210; adding aset of inputs to the reaction vessel S220; repeating one or moreprocesses (e.g., S230 through S250); and/or any other suitableprocess(es).

2. Benefits

The system and/or method can confer several benefits over conventionalsystems and methods buoyant particle processing.

First, in some variants, the system and/or method can confer the benefitof producing and/or modifying buoyant particles, thereby configuringthem for separating and/or isolating any or all of: cells, bacteria,viruses, exosomes and vesicles, molecules, DNA, RNA, or any othersuitable particle(s) from a suspension or solution. In a set of specificexamples, for instance, a set of buoyant particles are produced whichserve as an affinity support for molecular separation.

Second, in some variants, the system and/or method can confer thebenefit of producing and/or modifying buoyant particles, therebyconfiguring them for any or all of: animal, plant, and microbialculture. In a set of specific examples, for instance, a set of buoyantparticles are processed such that a surface of the buoyant particle actsas a buoyant platform during any or all of the cell culture andproliferation process.

Third, in some variants, the system and/or method can confer the benefitof producing and/or modifying buoyant particles, thereby configuringthem to provide a chemical and/or physical stimulus to a cell throughdirect contact and/or through elution of the stimulus into a solution.

Fourth, in some variants, the system and/or method can confer thebenefit of producing and/or modifying buoyant particles, therebyconfiguring them to serve as and/or display a chemical catalyst for usein the catalysis of chemical reactions.

Fifth, in some variants, the system and/or method can confer the benefitof minimizing and/or preventing buoyant particle breakage within anautomated and/or semi-automated buoyant particle processing system(e.g., manufacturing system). In a set of specific examples, forinstance, a system for processing buoyant particles includes a diaphragmpump (e.g., quaternary 4-piston diaphragm pump) to circulate a set ofbuoyant particles throughout the system, which can minimize a number ofbuoyant particles which are crushed while passing through the pump(e.g., in comparison with a peristaltic pump).

Sixth, in some variants, the system and/or method can confer the benefitof enabling an efficient and scalable filtering of buoyant particles(e.g., from debris, from a wash solution, from a buffer, from a subsetof relatively small buoyant particles, from a subset of relatively largebuoyant particles, etc.). In a set of specific examples, for instance, asystem for processing buoyant particles includes a hollow fiber moduleconfigured for tangential flow filtering, which functions to separatebuoyant particles from a remainder of a solution with minimal cloggingof the system due to buoyant particle flotation and the flow of theremaining solution.

Seventh, in some variants, the system and/or method can confer thebenefit of enabling any or all of: a semi-automated, fully automated,scalable, and large-scale system and method for buoyant particleprocessing. In a set of specific examples, for instance, the systemand/or method confer the benefit of constantly and consistentlycirculating (e.g., through a set of pumps, a tangential flow filter, anda prescribed set of flow parameters) a set of buoyant particles during aset of processes, wherein the circulation prevents clogging whileperforming the prescribed set of processes (e.g., washes, chemicaladditions, particle etching, filtering, etc.).

Eighth, in some variants, the system and/or method can confer thebenefit of minimizing user input and/or user performance in any or allof buoyant particle processing. In a set of specific examples, a set ofcentrifuging steps used in conventional mixing processes are replacedwith automated buoyant particle circulation and stirring. In another setof specific examples, the system can be partially or fully closed, whichcan eliminate or at least minimize required user intervention. In analternative variant, the system can interface with a centrifuge and/orthe method can include one or more centrifugation processes. In aspecific example, for instance, one or more materials (e.g., linkers)are processed with a centrifuge prior to entering the system 100.

Additionally or alternatively, the system and/or method can confer anysuitable benefit over conventional systems and methods for buoyantparticle processing.

3. System 100

The system 100 functions to produce a processed set of buoyantparticles. Additionally or alternatively, the system 100 can function tominimize and/or eliminate breakage of a set of buoyant particles duringbuoyant particle processing; minimize and/or eliminate user interventionduring buoyant particle processing (e.g., in comparison withconventional buoyant particle processing systems); enable a scaling upof a buoyant particle processing (e.g., prevent clogging of buoyantparticles); enable the production of a uniform (e.g., in surfacemodification, in size, etc.) set of buoyant particles; and/or performany other suitable function.

The system 100 receives as an input a set of buoyant particles.Additionally or alternatively, the system 100 can receive other inputs,such as a set of processing materials, which can include—but is notlimited to including—any or all of: chemicals (e.g., functional groups,linkers, etc.), proteins, buffers, reagents, washes, and/or any othersuitable materials for maintaining, modifying, or otherwise interactingwith the set of buoyant particles.

Each of the set of buoyant particles (equivalently referred to herein assubstrates) is preferably a microbubble (e.g., having a micron-scalediameter, having a diameter less than 1000 microns, having a diameterless than 100 microns, having a diameter between 10 and 100 microns,having a diameter of 50 microns, etc.), but can additionally include ananobubble (e.g., having a nanometer-scale diameter, having a diameterless than 1 micron, etc.), and/or any other suitable set of buoyantparticles. The set of buoyant particles received as an input to thesystem 100 are further preferably raw (e.g., unprocessed, absent ofsurface modifications, only washed, etc.) or partially processed (e.g.,glass coated in silane, glass coated in plastic, having a first surfacemodification, having a subset of surface modifications, polished,etched, etc.), but can additionally or alternatively be processed in anyother suitable way to any suitable degree. In examples, the buoyantparticles have a diameter between 10 nm and 100 nm (e.g., for use intargeting analytes in a subsequent protocol). In other examples, thebuoyant particles have a diameter between 1 μm and 30 μm (e.g., for usein targeting cells in a subsequent protocol); however, the particles canhave any other suitable dimension (e.g., diameter configured to enablethe buoyant particles to be included in the retentate of a tangentialflow filter as described below).

The set of buoyant particles (e.g., beads, spheres, micelles,microbubbles) can include any one or more of: plastic beads (e.g.,polypropylene beads, polyethylene beads, etc.), glass beads, lipid beads(e.g., stabilized liposome-based beads), hollow beads, solid beads,liquid-filled beads, gas-filled beads, and any other suitable type ofparticle.

The set of buoyant particles are preferably characterized by a firstdensity lower than that of the density (i.e., a second density) ofsurrounding fluids (e.g., buffers, solvents, fluids ranging from 0.1g/cm³ and 0.99 g/cm³, etc.). As such, the buoyant particles arepreferably configured to float when placed within the surrounding fluidsand/or fluids in a subsequent separation protocol, such as a separationprotocol described in U.S. application Ser. No. 16/004,874, filed 11Jun. 2018, which is incorporated herein in its entirety by thisreference. However, buoyant particles can alternatively be configuredwith any other suitable density relative to that of the other inputs tothe system.

In one variation, the set of buoyant particles includes microbubbles(e.g., gas-filled microparticles, hollow microspheres, colloidalbubbles) that can be spheroidal, skirted, ellipsoidal or any othersuitable three-dimensional shape. The shape of the microbubbles can varydynamically in response to the fluid dynamics of surrounding solutions(e.g., changing from one shape to another dictated by gravity,viscosity, and surface tension), but can alternatively be a fixed shape.In a specific example, the microbubbles are composed of borosilicateglass that can include a particle shell surrounding a particle core(e.g., gas filled, fluid-filled, particle-filled, etc.). However, theparticle shell can be alternatively composed of any other suitablematerial including lipids, proteins, surfactants, polymers, and/or anysuitable combination thereof. In this example, the glass microbubblescan be fabricated with a fixed spheroidal shape defining a particlediameter (e.g., ranging from between 5 to 30 micron), and a particleshell thickness (e.g., less than 2 micron thick). However, the buoyantparticles can be of any other suitable composition, shape, density,and/or dimension.

The system 100 produces as an output a set of processed buoyantparticles, wherein the processed buoyant particles refer to the inputbuoyant particles having undergone one or more surface modifications(e.g., through method 200 described below). The surface modificationspreferably include the application of one or more layers (e.g.,chemistries) applied to the buoyant particle surface, wherein the layersinclude any or all of: molecules, chemicals, moieties, proteins, organicmaterials, inorganic materials, protective shells, or any other suitablematerials. In some variations, for instance, the layers are formed bythe solution-based sequential addition of molecules onto the bubble.Additionally or alternatively, surface modifications can be applied byany or all of: in situ polymerization, chemical vapor deposition,polymeric coating in the presence of a solvent, polymeric coating in theabsence of a solvent, etching, or otherwise modifying the surface of theinput set of buoyant particles.

Each of the surface modifications (e.g., layers) preferably functions tofacilitate binding, such as any or all of: binding with the buoyantparticle surface (e.g., for a 1^(st) layer as described below), bindingwith a previously applied (e.g., lower) surface modification (e.g., fora 2^(nd) layer to bind with a 1^(st) layer, for a 3^(rd) layer to bindwith a 2^(nd) layer, etc.), binding with a subsequently applied (e.g.,above) surface modification (e.g., for a 1^(st) layer to bind with a2^(nd) layer, for a 2^(nd) layer to bind with a 3^(rd) layer, etc.),binding with a target material (e.g., solution, compound, material,cell, etc.) in subsequent applications (e.g., cell separation), or anyother suitable binding. Additionally or alternatively, any or all of thesurface modifications can function to prevent binding (e.g., 2^(nd)layer having a long chemical chain configured to prevent binding betweenthe 1^(st) and 3^(rd) layers, 2^(nd) layer having a long chemical chainconfigured to prevent binding between the 3^(rd) layer and the buoyantparticle surface, etc.), enhance buoyant particle longevity (e.g., shellto prevent breakage), prevent buoyant particle leeching (e.g., shell toprevent leeching on buoyant particle inner contents), and/or perform anyother suitable function.

In some variations, the processed buoyant particles include buoyantparticles functionalized with moieties for binding to a targetconstituent (e.g., red blood cells, white blood cells, T-cells,circulating tumor cells, stem cells, circulating nucleic acids, etc.)and can include any one or more of: charge-based moieties, nucleicacid-targeting moieties, protein-based moieties (e.g., cell adhesionmolecules, growth factors, synthetic proteins), and any other suitablemoiety. In a specific example, a particle shell of glass microbubblescan be coated with an aminosilane layer to allow for subsequent surfacefunctionalization with biomolecules (e.g., antibodies, aptamers,lectins, oligos, molecular barcodes, etc.). After glass microbubbleshave been amino-functionalized, the glass microbubbles are preferablycrosslinked to streptavidin. However, any other suitable chemicalprocedure can be performed for surface functionalization of thesubstrates (e.g., PEGylation, click chemistry, layer-by-layer assembly,ink-jet printing etc.) for selective capture of target constituents,using any other suitable moiety. The buoyant particles can additionallyand/or alternatively function as a signal delivery agent to targetconstituents (e.g., via a recombinant molecule bound to the surface ofthe substrate particle). In a specific example, CD3+T cells can becaptured using a microbubble displaying Cd28, a protein which canstimulate the T cell (e.g., inducing cell proliferation and cytokineproduction), a primary step to manufacturing T cells expressing achimeric antigen receptor (e.g., CAR-T cells) used in cell therapy(e.g., cancer treatment). However, the substrates can be otherwiseconfigured with any other suitable moiety for multifunctionalapplications including target-bound complex separation and extraction.

Each of the set of processed buoyant particles preferably includes a1^(st) layer (e.g., including one or more moieties as described above),equivalently referred to herein as the base layer, which functions tobind with the buoyant particle surface. Additionally or alternatively,the 1^(st) layer can function to prevent nonspecific binding bypreventing binding between subsequent layers and the buoyant particlesurface (e.g., by uniformly coating the buoyant particle surface). The1^(st) layer interfaces with (e.g., is applied to, layered on,functionalized with, etc.) the input buoyant particle surface (e.g., rawsurface, silane surface, etc.). Additionally or alternatively, the1^(st) layer can interface with a subsequent layer (e.g., 2^(nd) layeras described below, 3^(rd) layer as described below, 4^(th) layer,5^(th) layer, etc.), a target material for capture in a separationprocess, and/or any other suitable material(s).

The 1^(st) layer is preferably covalently bound to the buoyant particlesurface and configured for uniform particle coating (e.g., preventingpatchiness in each particle coating, uniformly coating all of the inputset of buoyant particles, etc.). The 1^(st) layer is further preferablyapplied as a uniform monolayer coating as opposed to nonuniformpolymeric coating (e.g., a carpet layer coating).

For glass (e.g., glass with a silane surface) buoyant particles, the1^(st) layer preferably includes one or more amino groups. In a specificexample, an aminosilane group of the 1^(st) layer interacts with ahydroxyl group on the surface of a glass buoyant particle. Additionallyor alternatively, the 1^(st) layer can include any suitable groups orother components.

The 1^(st) layer is preferably composed of multiple subcomponents (e.g.,chemical compounds, proteins, molecules, etc.) linked together, whichcan each have a different function and/or target (e.g., 1^(st)subcomponent is configured to bind with the buoyant particle surface and2^(nd) subcomponent is configured to bind with a subcomponent of a2^(nd) layer). Alternatively, the 1^(st) layer can include a singlesubcomponent, the multiple subcomponents can have the same functionand/or target, and/or the 1^(st) layer can have any other suitablestructure.

In some variations, the 1^(st) layer includes a first subcomponent,herein referred to as the “A element,” connected (e.g., linked, bonded,etc.) to a second subcomponent, herein referred to as the “B element,”wherein the A element is configured to bind (e.g., covalently, in areaction occurring in boiling toluene, etc.) with a surface of thebuoyant particle, and wherein the B element is configured to bind withany or all of: a subsequent layer (e.g., 2^(nd) layer, 3^(rd) layer,etc.), a target element (e.g., target molecule, target cell, etc.), orany other suitable component.

In a first specific example (e.g., as shown in FIG. 4), the A elementincludes the trimethoxysilane portion of 3-aminopropyltrimethoxysilane,which is configured to covalently bind with the buoyant particle surface(e.g., silane coated glass), and the B element includes the3-aminopropyl portion of 3-aminopropyltrimethoxysilane, which isconfigured to bind with a 2^(nd) layer (e.g., as described below).

Each of the set of processed buoyant particles can optionally include a2^(nd) layer (e.g., in combination with the 1^(st) layer, in absence ofa 1^(st) layer, etc.), equivalently referred to herein as the linkerlayer, which is preferably configured to bind with the 1^(st) layer(e.g., a B element of the 1^(st) layer). The linker layer canadditionally or alternatively be configured to bind with a subsequentlayer (e.g., an F element of a 3^(rd) layer), a target element, or anyother suitable material. The 2^(nd) layer preferably functions toincrease a distance between a 1^(st) layer (and/or the buoyant particlesurface) and a subsequent layer (and/or a target element), such as a3^(rd) layer (e.g., as described below). This can additionally oralternatively function to prevent binding between a subsequent layerwith a 1^(st) layer (and/or the buoyant particle surface), preventbinding between a target element and a previous layer (and/or thebuoyant particle surface), facilitate capture of a target element (e.g.,for a 2^(nd) layer having a capture molecule), and/or perform any othersuitable function. Further additionally or alternatively, the 2^(nd)layer (e.g., a D element of the 2^(nd) layer as described below) can beconfigured to be cleavable (e.g., to permit analyte release and/or cellrelease following capture). As such, one or more subcomponents (e.g.,2^(nd) subcomponent, 1^(st) subcomponent, 3^(rd) subcomponent, etc.) caninclude any or all of: disulfide bonds susceptible to chemicalreduction, polysaccharide chains susceptible to glycosidase digestion,polypeptide chains susceptible to peptidase digestion, DNA chainssusceptible to endonucleases (e.g., restriction-type endonucleases),and/or any other suitable materials and/or features. Additionally oralternatively, any other suitable layer can be configured to becleavable.

The 2^(nd) layer preferably interfaces with (e.g., is applied to,layered on, functionalized with, bound to, etc.) the 1^(st) layer (e.g.,a B element of the 1^(st) layer, an A element of the 1^(st) layer,etc.). Additionally or alternatively, the 2^(nd) layer can interfacewith any or all of: a subsequent layer (e.g., a 3^(rd) layer, a 4^(th)layer, etc.), a target element, the buoyant particle surface, or anyother suitable material.

In preferred variations, there is minimal or no binding between the2^(nd) layer and the buoyant particle surface (e.g., enabled by auniform coating of the buoyant particle surface by the 1^(st) layer,enabled by a choice of a B element of the 2^(nd) layer, etc.).Alternatively, binding can occur (e.g., accidentally, in regions havinga sparse distribution of a 1^(st) layer, etc.).

The 2^(nd) layer is preferably composed of multiple subcomponents (e.g.,chemical compounds, proteins, molecules, etc.) linked (e.g., bound)together, which can each have a different function and/or target (e.g.,one subcomponent is configured to bind with a subcomponent of the 1^(st)layer and another subcomponent is configured to bind with a subcomponentof a subsequent layer). Alternatively, the 2^(nd) layer can include asingle subcomponent, the multiple subcomponents can have the samefunction and/or target, and/or the 2^(nd) layer can have any othersuitable structure.

In some variations, the 2^(nd) layer includes a first subcomponent,herein referred to as the “C element,” connected (e.g., linked, bonded,etc.) to a second subcomponent, herein referred to as the “D element,”which is connected to a third subcomponent, herein referred to as the “Eelement.” The C element is preferably configured to bind (e.g.,covalently) with a B element of the 1^(st) layer and the D element; andthe E element is configured to bind with the D element and a subsequentlayer (e.g., 3^(rd) layer, 4^(th) layer, etc.). Additionally oralternatively, the 2^(nd) layer can be configured to bind with a targetelement (e.g., target molecule, target cell, etc.), and/or any othersuitable component.

In additional or alternative variations, the 2^(nd) layer can includetwo subcomponents (e.g., a C element connected to an E element), asingle subcomponent, additional subcomponents, and/or any suitablenumber of subcomponents arranged in any suitable way.

In a first specific example of the 2^(nd) layer, the C element includesan amino-reactive group (e.g., N-hydroxysuccinimide [NHS] ester)configured to bind (e.g., under predetermined conditions) to an aminogroup (e.g., B element) of the 1^(st) layer; the D element (e.g.,polyethylene glycol, polyethylene glycol chain, etc.) includes aninterposed region of a predetermined length (e.g., 12 repeat units, 24repeat units, less than 12 repeat units, greater than 12 repeat units,less than 30 repeat units, etc.), the D element configured to bind tothe C element and the E element; and the E element includes athiol-reactive group (e.g., maleimide) configured to bind with asubsequent layer (e.g., F element of a 3^(rd) layer as described below)and/or a target material.

Each of the set of processed buoyant particles can optionally include a3^(rd) layer (e.g., in combination with the 1^(st) and 2^(nd) layers, inabsence of one or both of the 1^(st) and 2^(nd) layers, etc.),equivalently referred to herein as the outer functional group, which ispreferably configured to bind with the 2^(nd) layer (e.g., an E elementof the 2^(nd) layer). The linker layer can additionally or alternativelybe configured to bind with a subsequent layer (e.g., a 4^(th) layer),the 1^(st) layer, the buoyant particle surface, a target element, or anyother suitable material. The 3^(rd) layer preferably functions to bindwith (e.g., and therefore capture) a target material. Additionally oralternatively, the 3^(rd) layer can function to prevent binding betweenthe target material and a previous layer and/or perform any othersuitable function.

The 3^(rd) layer preferably interfaces with (e.g., is applied to,layered on, functionalized with, bound to, etc.) the 2^(nd) layer (e.g.,an E element of the 2^(nd) layer, a D element of the 2^(nd) layer, a Celement of the 2^(nd) layer, etc.). Additionally or alternatively, the3^(rd) layer can interface with any or all of: a subsequent layer (e.g.,a 4^(th) layer, a 5^(th) layer, etc.), an intermediate layer arrangedafter the 2^(nd) layer, a target element, the buoyant particle surface,or any other suitable material.

In preferred variations, there is minimal or no binding between the3^(rd) layer and the buoyant particle surface (e.g., enabled by auniform coating of the buoyant particle surface by the 1^(st) layer,enabled by a choice of an F element of the 3^(rd) layer, etc.) as wellas minimal or no binding between the 3^(rd) layer and the 1^(st) layer.Alternatively, binding can occur (e.g., accidentally, in regions havinga sparse distribution of a 1^(st) layer, etc.).

The 3^(rd) layer is preferably composed of multiple subcomponents (e.g.,chemical compounds, proteins, molecules, etc.) linked (e.g., bound)together, which can each have a different function and/or target (e.g.,one subcomponent is configured to bind with a subcomponent of the 2^(nd)layer another subcomponent is configured to bind with a targetmaterial). Alternatively, the 3^(rd) layer can include a singlesubcomponent, the multiple subcomponents can have the same functionand/or target, and/or the 3^(rd) layer can have any other suitablestructure.

In some variations, the 3^(rd) layer includes a first subcomponent,herein referred to as the “F element,” connected (e.g., linked, bonded,etc.) to a second subcomponent, herein referred to as the “G element.”The F element is preferably configured to bind (e.g., covalently) withan E element of the 2^(nd) layer and the G element is configured to bindwith a target element (e.g., target molecule, target cell, etc.).Additionally or alternatively, the 3^(rd) layer can be configured tobind with a subsequent (e.g., 4^(th) layer, 5^(th) layer, etc.), the1^(st) layer, a surface of the buoyant particle, and/or any othersuitable component.

In additional or alternative variations, the 3^(rd) layer can include asingle subcomponent, more than two subcomponents, and/or any suitablenumber of subcomponents arranged in any suitable way.

In a first specific example of the 3^(rd) layer, the F element includesa thiol group configured to bind to a thiol-reactive group (e.g., Eelement) of the 2^(nd) layer, and the G element includes a captureelement (e.g., capture molecule, capture group, streptavidin, one ormore antibodies, a lectin, an oligonucleotide sequence, etc.) configuredto bind with a target material (e.g., a biotinylated species).

The processed buoyant particle can additionally or alternatively includeany number of surface modifications arranged (e.g., layered) in anysuitable way. In some variations, additional or alternative to thosedescribed above, the processed buoyant particle includes any or all of:one or more proteins (e.g., polymerized glycidol) which can function,for instance to increase a surface roughness of the buoyant particle(e.g., and therefore improve a binding ability of the buoyant particle);a shell (e.g., polymer shell) which can function, for instance, toprevent leeching of one or more components of the buoyant particle(e.g., glass buoyant particle) into the surrounding solution; and achange in charge (e.g., applied charge, induced charge, switched charge,etc.) of the buoyant particle surface which can function, for instance,to enable DNA capture (e.g., based on charge switching of silica).

In a first variation (e.g., as shown in FIG. 3, as shown in FIGS. 5A-5D,etc.), the processed buoyant particle includes a 1^(st) layer bound tothe buoyant particle surface, a 2^(nd) layer bound to the 1^(st) layer(e.g., to a B element of the 1^(st) layer), and a 3^(rd) layer bound tothe 2^(nd) layer (e.g., to an E element of the 2^(nd) layer). In aspecific example, the processed buoyant particle includes a 1^(st) layerof 3-aminopropyltrimethoxysilane bound to an amino group; a 2^(nd) layerof an amino-reactive group configured to bind with the amino group ofthe 1^(st) layer, a polyethylene glycol chain bound to theamino-reactive group, and a thiol-reactive group bound to thepolyethylene glycol chain; and a 3^(rd) layer of a thiol groupconfigured to bind with the thiol-reactive group of the 2^(nd) layer,and a capture molecule.

In a second variation (e.g., as shown in FIGS. 6A-6C), the processedbuoyant particle includes a 1^(st) layer bound to the buoyant particlesurface and a 3^(rd) layer bound to the 1^(st) layer.

In a third variation, the processed buoyant particle includes a buoyantparticle having a surface with an altered charge density.

3.1 System: Reaction Vessel 110

The system 100 includes a reaction vessel 110, equivalently referred toherein as a process chamber, which functions to contain the set ofbuoyant particles and any suitable solutions (e.g., buffers, solvents,etc.) during one or more processing steps (e.g., while coated in one ormore layers, while etched, etc.). Additionally or alternatively, thereaction vessel 110 can function to enable an even coating of each ofthe set of buoyant particles, such as by having a size above apredetermined threshold such that the buoyant particles can have aparticle-to-particle spacing above a predetermined threshold, maintain aproper environment (e.g., temperature, humidity, etc.) for buoyantparticle processing (e.g., heating the set of particles, boiling asolution, cooling the set of particles, etc.), and/or perform any othersuitable function.

The reaction vessel 110 receives the set of buoyant particles (e.g.,input set of buoyant particles, partially processed buoyant particles,processed buoyant particles) and/or any other suitable solutions andmaterials, such as those used in processing the buoyant particles (e.g.,materials of 1^(st) layer, materials of 2^(nd) layer, materials of3^(rd) layer, buffers, reagents, etc.). The reaction vessel no canreceive the set of buoyant particles from any or all of: a user (e.g.,user dispensing input buoyant particles into the reaction vessel), areservoir (e.g., through an automated mechanism such as an automatedcirculation subsystem of the system), another component of the system(e.g., from a filter through a set of fluid pathways as describedbelow), and/or from other suitable individual or system component. Insome variations, the set of input buoyant particles are received into acavity of the reaction vessel from a first source (e.g., user, chamber,etc.) and subsequently received after each of a set of processing stepsfrom another component of the system (e.g., through a set of pathways ina closed system).

The reaction vessel can receive any number of solutions (e.g., buffersolutions includes at least one of PBS, BSA, and/or EDTA), which caninclude any suitable reagents, growth factors, chemical compounds,solvents, and/or be of any suitable pH, temperature, or othercharacteristic to support the viability of buoyant particles (e.g.,minimize particle aggregation, improve long-term storage, etc.),processing materials, and/or target constituents.

The reaction vessel 110 is preferably made of glass but can additionallyor alternatively be made of a polymer (e.g., plastic), metal, wood, orany other suitable material. The reaction vessel preferably defines asingle cavity, such that the set of buoyant particles are processed inthe same environment, but can additionally define multiple cavities(e.g., to scale up a method as described below). Furthermore, anysurface (exterior and/or interior) of the reaction vessel 110 can beoptionally treated with a surface coating (e.g., to influence surfaceproperties, adhesion properties, optical properties, to prevent adhesionof the set of buoyant particles to an inner surface of the reactionvessel, etc.).

The reaction vessel 110 can include and/or define a set of one or moreinlets 112, which function to receive any or all of: the set of buoyantparticles (e.g., the input set of buoyant particles, buoyant particleshaving one or more surface modifications, etc.), buffers (e.g., washbuffers), reagents, processing materials (e.g., 1^(st) layer materials,2^(nd) layer materials, 3^(rd) layer materials, etc.), a stirringsubsystem (e.g., as described below), and/or any other suitablematerials. The set of inlets 112 preferably includes multiple inlets(e.g., each configured to receive a different material), furtherpreferably a set of inlets arranged on a superior (e.g., top) surface ofthe reaction vessel 110, but can additionally or alternatively include asingle inlet and/or a set of inlets arranged at any suitable surface ofthe reaction vessel 110. In one variation (e.g., as shown in FIG. 9),the reaction vessel 110 includes a first inlet configured to receive awash buffer (e.g., from a wash buffer chamber), a second inletconfigured to receive a set and/or sets of processing materials (e.g.,1^(st) layer materials, 2^(nd) layer materials, 3^(rd) layer materials,etching materials, etc.), and a third inlet configured to receive thebuoyant particles (e.g., from a filter as described below) after one ormore surface modification processes and/or washes.

The reaction vessel 110 can additionally include and/or define a set ofone or more outlets 114, which functions to remove any or all of thecontents of the reaction vessel no (e.g., after a surface modificationprocess, after a wash, etc.). The reaction vessel 110 preferablyincludes a single outlet 114 arranged on an inferior (e.g., bottom)surface of the reaction vessel no, but can additionally or alternativelyinclude a single outlet and/or multiple outlets arranged on any suitablesurface of the reaction vessel. In one variation (e.g., as shown in FIG.9), the reaction vessel no includes an outlet configured to remove thecontents of the reaction vessel 110 (e.g., which are next transported toa feed inlet of the filter) after a surface modification process suchthat the reaction vessel 110 can be prepared with new materials (e.g.,from any or all of the set of inlets described above) for a subsequentprocess.

The reaction vessel can optionally include and/or be configured tointerface with any or all of: a set of reservoirs and/or chamberscontaining one or more materials (e.g., buffers, solutions, wash buffer,surface modification materials, layer materials, buoyant particles,etc.); a heating and/or cooling subsystem (e.g., to achieve and/ormaintain a temperature required for processing the buoyant particles); asensor system (e.g., temperature sensor, pressure sensor, flow ratesensor, etc.); and/or any other suitable component(s) configured toenable a processing of the set of buoyant particles.

In a first variation (e.g., as shown in FIG. 9), the reaction vesselincludes a 1^(st) inlet configured to receive (e.g., fluidly connectedto) a wash buffer (e.g., from a wash buffer reservoir), a 2^(nd) inletconfigured to receive a set of processing materials, a 3^(rd) inlet toreceive the set of buoyant particles (e.g., after being filtered), andan outlet configured to receive the contents of the reaction vessel(e.g., and pass them through a filter using a pump). Additionally oralternatively, the system can include any or all of: a 4^(th) inletconfigured to receive a stirring mechanism, multiple outlets, any numberof additional inlets, and/or any other suitable components.

3.2 System: Stirring Mechanism 120

The system 100 includes a stirring mechanism 120, which functions toconstantly and consistently circulate the set of buoyant particles,which can in turn function to prevent and/or minimize clogging of one ormore system components, to prevent the buoyant particles from floatingand aggregating at the top of the reaction vessel (e.g., therebyexperiencing an overall uneven coating of processing materials).Additionally or alternatively, the stirring mechanism 120 can functionto mix a set of buoyant particles with a set of processing materials(e.g., to ensure even and thorough coating of buoyant particles with aset of surface layers), perform the method in absence of centrifugation(e.g., as performed in conventional buoyant particle processing, asperformed in small-scale buoyant particle processing, etc.), and/orperform any other suitable function.

The stirring mechanism 120 preferably includes an impeller arranged in acavity of the reaction vessel (e.g., such that the impeller is fullysubmerged by a processing solution, proximal to a bottom surfacedefining the internal cavity, etc.) and a motor (e.g., electric motor)configured to rotate the impeller. The stirring mechanism 120 canadditionally or alternatively include and/or be configured to interfacewith any or all of: a power source (e.g., battery, wall outlet, etc.)configured to power the electric motor, a rod (e.g., a stir rod, rodconnecting the motor to the impeller, etc.), any suitable stirringdevice (e.g., fanned rod, magnetic stir bar, etc.), and/or any otherstirring device.

The stirring mechanism 120 is preferably operated without or withminimal user intervention (e.g., turned “on” and “off” by a user) butcan additionally or alternatively be manually operated. The stirringmechanism 120 preferably stirs the contents of the reaction vessel at aspeed fast enough to maintain an approximately uniform spacing betweenthe buoyant particles, such that each buoyant particle is coated withthe processing materials, yet slow enough to prevent and/or minimize abreakage of the buoyant particles. Additionally or alternatively, theimpeller can rotate at any suitable speed(s).

In a first variation of the stirring mechanism 120, the stirringmechanism 120 includes an impeller arranged proximal to a bottom surfacedefining an internal cavity of the reaction vessel, wherein the impelleris rotated by an electric motor arranged above the reaction vessel andcoupled to a power source (e.g., battery, wall outlet, etc.). In aspecific example of this variation, the angular speed of the impeller isbetween 2400 and 3000 °/s (and/or between 400 and 500 rpm). Additionallyor alternatively, an angular speed of the impeller can be less than 2400°/s, greater than 3000 °/s, less than 400 rpm, greater than 500 rpm,and/or have any other suitable value or range of values.

3.3 System: Set of Pumps 130

The system 100 includes a set of one or more pumps 130, which canindividually and/or collectively function to transport any or all of:the set of buoyant particles, one or more solutions (e.g., buffers,reagents, etc.), processing materials, and/or any other material(s)throughout the system 100 (e.g., within components, between components,into the system, out of the system, etc.). One or more of the set ofpumps 130 further preferably functions to prevent and/or minimizebreakage of the set of buoyant particles, but the set of pumps 130 canadditionally or alternatively perform any other suitable function.

The set of pumps 130 includes a first pump 132, which is arrangeddownstream of an outlet of the reaction vessel 110 and upstream of afilter (e.g., as described below). The first pump 132 is preferablyconnected through a fluidic pathway (e.g., tubing) to an outlet (e.g.,inferior outlet) of the reaction vessel 110, but can additionally oralternatively be coupled to the reaction vessel 110 in any othersuitable way. The first pump 132 functions to transport the set ofbuoyant particles from the reaction vessel no to the filter and toprevent and/or minimize breakage of the set of buoyant particles duringtransport. The first pump 132 preferably includes a diaphragm pump(e.g., 4-piston diaphragm pump, 2-piston diaphragm pump, etc.), whereinthe diaphragm pump enables minimal contact (e.g., in comparison withanother type of pump, in comparison with a peristaltic pump, etc.) andminimal associated contact forces (e.g., contact force large enough tobreak a buoyant particle) between the pump and the particles.Additionally, the diaphragm pump can enable minimal contact and minimalassociated contact forces between adjacent particles (e.g., whenrestricted in a small diameter passageway of a peristaltic pump).

The first pump 132 is preferably operated in accordance with a flow rateconfigured to minimize breakage of buoyant particles. The flow rate ofthe first pump 132 (and/or a second pump 134) is further preferablydetermined based on any or all of: a total length of the system (e.g.,distance particle traverses from the reaction vessel to the filter andback to the reaction vessel), a timing of one or more processes of themethod (e.g., time required to add a new set of inputs to the reactionvessel such that the buoyant particles aren't placed into an emptyreaction vessel, etc.). In a variation, the first pump 132 is operatedwith a flow rate between 400 mL/min and 600 mL/min. Additionally oralternatively, the first pump 132 can be operated with a flow ratebetween 200 mL/min and 1000 mL/min, with a flow rate less than 400mL/min, with a flow rate greater than 600 mL/min, and/or in accordancewith any suitable operating parameters having any suitable values.

The set of pumps 130 preferably includes a second pump 134, wherein thesecond pump 134 functions to pump one or more fluids (e.g., buffers,solvents, washes, etc.) into the reaction vessel 110. The second pump134 preferably does not interact with the set of buoyant particles, butcan additionally or alternatively pump buoyant particles into thereaction vessel, remove any suitable materials from the reaction vessel110, transport one or more solutions between components of the system,or otherwise interact with any suitable materials of the system 100. Thesecond pump 134 is preferably arranged upstream of the reaction vessel110 and connected to the reaction vessel through a fluidic pathway(e.g., flexible tube) connected to a superior inlet of the reactionvessel 110. Additionally or alternatively, the second pump 134 can bearranged downstream of a wash buffer chamber (e.g., and configured topump fresh wash buffer into the system during washes), a containerholding a set of processing materials, and/or any other solutions andmaterials to be added to the reaction vessel no. The second pump 134 canbe a different pump type (e.g., peristaltic pump) than the first pump,the same pump type (e.g., diaphragm pump), or any other suitable pumptype.

The set of pumps 130 can additionally or alternatively include a singlepump, additional pumps, a different pump type, or any other suitablepumps in any suitable arrangement.

In one variation of the set of pumps 130 (e.g., as shown in FIG. 9), theset of pumps includes a first pump 132 arranged between an outlet of thereaction vessel 100 and an inlet of a filter 140, and a second pump 134arranged between a wash buffer chamber and the reaction vessel 110. In aspecific example, the first pump 132 is a diaphragm pump (e.g.,quaternary diaphragm pump) configured to gently transport a set ofbuoyant particles from the reaction vessel 110 to the filter 140, andwherein the second pump 134 is a peristaltic pump configured to pumpwash buffer into and/or throughout the system (e.g., into the reactionvessel) during washes (e.g., in-between particle processing steps).

3.4 System: Filter 140

The system includes a filter 140, which functions to separate the set ofbuoyant particles from waste materials (e.g., wash buffers, processingmaterials, debris, etc.) after one or more buoyant particle processingprocesses. Additionally or alternatively, the filter 140 can function toseparate the set of buoyant particles from any other solutions andmaterials (e.g., separate from a storage buffer prior to a firstprocessing process); prevent and/or minimize a clogging of one or morecomponents of the system (e.g., materials with highest buoyancy passthrough the filter whereas other materials are collected in the filter);directly collect buoyant particles (e.g., from an outlet of the filter);enable a fast washing (e.g., less than 10 minutes, less than 5 minutes,between 5 seconds and 2 minutes, less than 5 seconds, etc.) of buoyantparticles; and/or perform any other suitable function.

Further additionally or alternatively, the filter 140 can function toenable large volume buoyant particle processing relative to conventionalprocessing protocols (e.g., including centrifugation, including manualprocessing and/or preparation, etc.). In some conventional processingmethods, for instance, a set of centrifugation processes are required toseparate the set of buoyant particles from surrounding solutions andmaterials. Conventional processing methods can require, for instance,any or all of: limited-volume, batched separation (e.g., based oncentrifuge tube volumes); user intervention (e.g., to operate thecentrifuge, to pipette the buoyant particles into and/or out ofcentrifuge tubes, to place and/or remove centrifuge tubes from thecentrifuge, etc.); removal of the buoyant particles from a system (e.g.,a closed system, a reaction vessel, etc.); and/or any otherrequirements. The filter 140 preferably functions to eliminate and/orminimize one or more of these requirements, but can additionally oralternatively eliminate and/or minimize another requirement, have one ormore of these requirements (e.g., be used in conjunction with acentrifuge, etc.), or perform any other suitable function.

The filter 140 preferably separates the set of buoyant particles from asurrounding solution, wherein the surrounding solution includes any orall of: one or more processing materials (e.g., 1^(st) layer elements,2^(nd) layer elements, 3^(rd) layer elements, etc.); one or more fluidssuch as buffers, solvents, other solutions in the reaction vessel 110;debris (e.g., broken buoyant particles); and/or any other suitablematerials. The buoyant particles are preferably separated from asurrounding solution after each particle processing process (e.g., andprior to a subsequent processing process), but can additionally oralternatively be separated from a surrounding solution prior to aprocessing process (e.g., to filter out a storage buffer); be separatedfrom a second set of buoyant particles (e.g., based on size such thatthe set of buoyant particles are uniformly sized); and/or be otherwiseseparated from any suitable material.

The filter 140 preferably includes a tangential flow filter (e.g.,hollow fiber membrane filter), wherein the set of buoyant particles(which form the retentate) are retained within the membrane (e.g.,semi-permeable membrane, wall, barrier, inner wall, inner diameter,outer wall, outer diameter, etc.) of (e.g., based on buoyancy, based onsize, based on buoyancy and size, etc.) a set of filter elements (e.g.,columns, fibers, plate having apertures, mesh, etc.), and wherein partor all of the remaining solution (e.g., permeate) passes through the setof filter elements (e.g., tangential to the flow of the feedchannel/retentate, non-parallel with respect to a central axis of eachfilter element, perpendicular to a central axis of each filter element,due to cross flow, etc.). The flow direction of the buoyant particles(which form the retentate) within the filter membrane(s) is non-parallel(e.g., perpendicular, approximately perpendicular, at an angle between70 and 100 degrees, etc.) with respect to the flow direction of theremaining solution (which forms the permeate) through the membrane(e.g., as shown in FIG. 7). In preferred variations of the filter 140,for instance, the filter 140 has a nonzero angle (e.g., 90 degrees, 45degrees, between 45 and 90 degrees, less than 45 degrees, greater than90 degrees, etc.) between a flow direction of the feed/retentate (e.g.,central axis of the filter element) and a flow direction of thepermeate.

Additionally or alternatively, the filter 140 can include a direct flowfilter (e.g., mesh filter, fret filter, filter as shown in FIG. 8,etc.), wherein an inlet of the filter is arranged perpendicular to aflow direction (e.g., direction of a central axis) within the filterelements.

The filter 140 can optionally be a gravity-assisted filter (e.g., directflow filter, filter wherein the flow direction of the remaining solutionthrough the filter is aligned or at least partially aligned withgravity, etc.), but can additionally or alternatively be assisted with apump (e.g., first pump 132), an attractive mechanism (e.g., magnetarranged downstream of a filter element to attract magnetic particles),unassisted, and/or otherwise assisted.

In a first variation (e.g., as shown in FIG. 7), the filter 140 is atangential flow filter arranged in a vertical (e.g., along the directionof gravity) or partially vertical (e.g., near vertical, non-horizontal,etc.) alignment with respect to gravity, wherein a feed inlet of thefilter 140 is parallel with a retentate outlet of the filter 140,wherein the set of buoyant particles travel from the feed inlet to theretentate outlet within a set of hollow fiber filter elements along adirection aligned with the central axis of each hollow fiber. A permeateoutlet of the filter 140 is arranged perpendicular to both the feedinlet and the retentate outlet, wherein the permeate (e.g., remainingsolution) exits through the semi-permeable membranes of each filterelement (e.g., through cross flow) and exits the filter at a flowdirection perpendicular to the central axis of the filter elements(e.g., the retentate flow direction, and feed flow direction, theretentate and feed flow directions, etc.).

In a second variation (e.g., as shown in FIG. 8), the filter 140 is adirect flow filter arranged in a vertical (e.g., along the direction ofgravity) or partially vertical (e.g., near vertical, non-horizontal,etc.) alignment with an inlet arranged perpendicular to this (e.g.,horizontal, near horizontal, etc.). An outlet of the filter can bearranged perpendicular to the inlet (e.g., to leverage a buoyancy of thebuoyant particles, with the assistance of a pump, etc.), parallel to theinlet (e.g., as shown in FIG. 8), and/or at any suitable angle withrespect to the inlet (e.g., between 0 and 90 degrees, greater than 90degrees, etc.). Additionally or alternatively, the filter 140 can bearranged in any suitable orientation with any suitable angles betweenfilter sub-components. In variations of the direct flow filter, the flowdirections of the retentate and the permeate are preferably parallel(e.g., retentate flow direction is parallel with the permeate flowdirection in FIG. 8). Alternatively, the flow directions of theretentate and permeate can be arranged with any suitable angle respectto each other (e.g., 45 degrees, 90 degrees, between 160 and 200degrees, less than 180 degrees, greater than 180 degrees, etc.). In aset of specific examples, the filter 140 is arranged with a nonzero tiltangle, such that the set of buoyant particles are directed toward anoutlet of the filter (e.g., arranged on a superior surface (top) of thefilter).

The filter 140 is preferably arranged downstream of the reaction vessel110, further preferably additionally downstream of a first pump 132, andupstream of an inlet of the reaction vessel 110, such that the filter140 receives the contents of the reaction vessel no (e.g., after aparticle processing process), which can be optionally brought to aninlet of the filter through the pump 132 (e.g., via a set of fluidicpathways). Additionally or alternatively, the filter 140 can be arrangedwith respect to other components of the system 100 in any suitable way.

Each filter element (e.g., channel, hollow fiber, tube, cylinder,aperture in a direct flow filter, pore in a direct flow filter, frit ina direct flow filter, etc.) of the filter preferably has a cross sectionwith a characteristic diameter (e.g., inner diameter, outer diameter,inner diameter of a hollow fiber, etc.) larger than the diameter of eachbuoyant particle (e.g., in a transverse flow filter), wherein thecharacteristic diameter defines an upper limit of a size range ofparticles which can enter the filter elements, such that the buoyantparticles can enter the filter elements (e.g., travel along a length ofa hollow fiber, pass through an aperture, enter an inner lumen of afilter element, etc.). The characteristic diameter of each filterelement is further preferably larger than the surrounding material(e.g., processing materials, debris, etc.) to be separated from thebuoyant particles, such that the surrounding materials can also enterthe filter elements. Additionally or alternatively, any or all of thefilter elements can have a characteristic diameter smaller than at leasta partial set of the buoyant particles (e.g., to filter the buoyantparticles by size, in a direct flow filter, etc.), a variable diameter(e.g., range of diameters among the filter elements), and/or any othersuitable dimensions. The filter elements are preferably identical (e.g.,have the same diameter, have the same dimensions, etc.), but canadditionally or alternatively have variation (e.g., an increasingdiameter from upstream to downstream filter elements such that thesurrounding material to be separated from the buoyant particles issorted by size).

In some variations (e.g., wherein each buoyant particle is amicrobubble), a characteristic diameter of each filter element (e.g.,each hollow fiber in a hollow fiber filter) is greater than 10 micronsand less than 10,000 microns (e.g., between 100 and 1000 microns,greater than 30 microns, less than 100 microns, greater than 100microns, etc.). Additionally or alternatively, the filter elements canhave various diameters (e.g., each greater than 10 microns, each between100 and 1000 microns, each between 10 and 30 microns, etc.), or anyother suitable diameter(s).

Each filter element further preferably includes and/or defines (e.g., inthe case of a tangential flow filter) a semi-permeable membrane having aset of membrane pores, which function to separate (filter) one or moresurrounding materials (e.g., through cross flow) from the buoyantparticles which remain within the membrane of the filter element. Adiameter of each of the set of membrane pores is preferably smaller thana diameter of the buoyant particle and larger than a diameter than anyor all of the components in the remaining solution (permeate). Themembrane pores define a central axis arranged in a non-parallel (e.g.,perpendicular, substantially perpendicular, at an angle between 80 and100 degrees, etc.) orientation with respect to a central axis of thefilter element (e.g., defining the characteristic diameter, innerdiameter of a hollow fiber, etc.). As such, the membrane pores functionto prevent buoyant particles from exiting through the semi-permeablemembrane while enabling the surrounding material (e.g., all of thesurrounding material, a portion of the surrounding material, etc.) toexit the filter element through the semi-permeable membrane (e.g.,through cross flow).

In some variations, the diameter of at least each of a partial set ofmembrane pores is less than 10 microns (e.g., 0.2 microns, 0.5 microns,between 0.1 and 1 microns, greater than 1 micron, etc.), but canadditionally or alternatively be less than 30 microns, greater than 10microns, between 10 and 100 microns, or have any other suitablediameter.

The length of the filter (e.g., length of each hollow fiber module) asmeasured in the direction of flow of the materials being collected byand/or within the filter elements can have any suitable value (e.g.,greater than 12 inches, less than 12 inches, between 4 inches and 18inches, between 10 inches and 30 inches, greater than 30 inches, lessthan 60 inches, etc.). The number of filter elements (e.g., hollowfibers, pores, etc.) in the filter is preferably configured to enable alarge-scale processing (e.g., enable a predetermined flow rate, enable apredetermined reaction vessel volume, enable a predetermined totalprocessing time, etc.) of buoyant particles (e.g., without clogging thefilter) but can additionally or alternatively configured.

For the set of filter elements (e.g., hollow fibers), there can be anonzero spacing between adjacent filter elements, can be in contactalong a partial length of adjacent filter elements, can be in contactalong a full length of adjacent filter elements, or otherwise arranged.

The filter can additionally include one or more ports, such as a portfor adjusting pressure in the filter, which can function to: keep thesolution flowing through the filter, assist and/or enable the set ofbuoyant particles (e.g., retentate) to exit the filter (e.g., andre-enter the reaction vessel), assist and/or enable the surroundingmaterials (e.g., permeate) to pass through the filter elements (e.g.,traverse a length of a hollow fiber module), unclog the filter, and/orperform any other suitable function.

The filter element preferably includes one or more plastic materials inthe semi-permeable membrane, which define a set of membrane poresthrough which the surrounding materials exit the filter elements.Additionally or alternatively, the filter elements can include any orall of: a polymer, glass, wood, metal, natural fiber, synthetic fiber,fabric, ceramic, and/or any other suitable material(s).

In one variation, the filter 140 includes a hollow fiber tangentialfilter, wherein the hollow fiber filter includes a bundle of hollowfibers (e.g., closely packed, with negligible gaps between adjacenthollow fibers, etc.). The hollow fiber filter receives the set ofbuoyant particles along with the surrounding solution (e.g., after aparticle processing process, after a wash step, etc.) from the reactionvessel 110 (e.g., after being passed through a first pump 132) andseparates the set of buoyant particles (e.g., retentate) from thesurrounding solution (e.g., wash buffer and debris), wherein the set ofbuoyant particles, which form the retentate, flows through the hollowfibers (e.g., in a direction parallel with a central axis of the hollowfibers) and exits through an outlet (e.g., and back into the reactionvessel 110, into a separate container, etc.) whereas the surroundingsolution, which forms the permeate, exits the hollow fibers through aset of fiber membrane pores (e.g., in a semi-permeable membrane) in across flow direction, wherein the cross flow direction is perpendicularto the retentate flow direction. The permeate can then optionally becollected at a waste chamber.

3.5 System: Set of Pathways 150

The system 100 includes a set of pathways 150, which can include anynumber of tubes (e.g., flexible tubes), channels, conduits, columns,and/or any other suitable pathways configured to transport the set ofbuoyant particles (e.g., and any surrounding solution) throughout thesystem 100. The set of pathways 150 further preferably functions toconnect multiple components of the system together (e.g., to create aclosed system), consistently and constantly circulate the inputs of thesystem (e.g., to prevent clogging), and/or perform any other suitablefunction.

The set of pathways 150 can include and/or define any or all of: a setof ports (e.g., to regulate a pressure within any or all of the system),a set of inlets, a set of outlets, a surface coating and/or surfacemodifications (e.g., to reduce friction within an inner lumen of apathway, to prevent attraction between the set of buoyant particles andan inner lumen of the pathway, etc.), and/or any other suitablefeatures.

In one variation, the set of pathways 150 includes a set of flexibletubes which connect adjacent components of the system 100 together,through which any or all of: the set of buoyant particles, buffers,processing materials, debris, reagents, and/or other solutions andmaterials circulate.

3.6 System: Additional Components

The system 100 can additionally or alternatively include any or all of:a set of sensors, such as a pressure sensor (e.g., a pressure sensor tomeasure an internal pressure of a closed system 100, a pressure sensorto measure a pressure within a fluidic pathway, etc.), a temperaturesensor (e.g., to maintain a predetermined temperature or range oftemperatures for buoyant particle processing), a flow rate sensor,and/or any other suitable sensor(s); a control subsystem (e.g.,processing subsystem, processor, controller, etc.); a power source(e.g., to power the stirring mechanism, to power one or more pumps,etc.); and/or any other suitable component(s).

3.7 System: Variations

In one variation of the system 100 (e.g., as shown in FIG. 9), thesystem 100 includes: a reaction vessel no, wherein the reaction vesselno functions to receive a set of buoyant particles (e.g., microbubbles),a set of processing materials (e.g., 1^(st) layer materials, 2^(nd)layer materials, 3^(rd) layer materials, etc.), and any additionalsolutions (e.g., buffers) or materials, wherein the set of buoyantparticles are at least partially processed within the reaction vessel110; a stirring mechanism 120 including an impeller arranged within acavity of the reaction vessel 110; a stir rod connected to the impeller;and an electric motor arranged above the reaction vessel 110 andconfigured to rotate the stir rod; a first pump 132 (e.g., a quaternarydiaphragm pump), wherein the first pump 132 is configured to transport(e.g., gently transport, transport with minimal breakage of the set ofbuoyant particles, etc.) the set of buoyant particles (e.g., and othercontents of the reaction vessel 110) to a tangential flow filter 140(e.g., hollow fiber filter), wherein the filter 140 collects (e.g., andcollects at a waste chamber) solutions and materials excluding the setof buoyant particles through a set of membrane pores of the filterelements (e.g., hollow fibers having an inner diameter larger than adiameter of each of the buoyant particles and a set of membrane poreswith a diameter smaller than a diameter of each of the set of buoyantparticles but larger than a diameter of the components of thesurrounding solution) and outputs the set of buoyant particles through aretentate outlet of the filter; and a set of fluidic pathways 150 whichcirculate the inputs of the system 100 (e.g., the set of buoyantparticles, solutions, processing materials, etc.) from the reactionvessel no to the filter 140 and back to the reaction vessel no or finalcollection container (e.g., after all processing processes have beencompleted). The system 100 can additionally include any or all of: afirst pump 130 (e.g., a peristaltic pump) configured to add one or moreinputs (e.g., wash buffer) into the reaction vessel no; a set ofpressure sensors (e.g., to monitor a pressure in one or more fluidicpathways, to monitor a pressure in the filter, to monitor a pressure inthe reaction vessel 110, etc.), and/or any other suitable components.

4. Method 200

The method 200 for buoyant particle processing functions to apply one ormore surface modifications to a set of buoyant particles. Additionallyor alternatively, the method 200 can function to wash a set of buoyantparticles, filter a set of buoyant particles (e.g., from broken particlefragments, debris, particles having a different size, etc.), preventbreakage of a set of particles, operate a closed system with minimaland/or no user intervention, and/or perform any other suitable function.

The method 200 is preferably performed with a system 100 as describedabove but can additionally or alternatively be performed with anysuitable system.

4.1 Method: Preprocessing a Set of Buoyant Particles S210

The method 200 can optionally include preprocessing the set of buoyantparticles S210 (e.g., prior to an application of a first layer), whichfunctions to prepare the set of buoyant particles S210 for any or all ofthe subsequent processes (e.g., application of a first layer) of themethod 200. S210 can include any or all of: the application of a surfacelayer (e.g., protective surface layer, protective shell to preventleaching, adhesion-promoting surface layer, etc.), the modification of abuoyant particle surface (e.g., etching, increasing surface roughness,etc.), or any other suitable process.

S210 is preferably performed first in the method 200 but canadditionally or alternatively be performed multiple times throughout themethod 200, later in the method 200 (e.g., after washing, afterfiltering, after filtering for size, etc.), not performed at all, orotherwise performed at any suitable time(s).

S210 can be performed within a system 100 (e.g., in a reaction vessel110), outside of the system 100 (e.g., at a microbubble manufacturingfacility, at a lab bench, etc.), or at any other suitable location.

In one variation, the method 200 includes S210, wherein S210 isperformed prior to the application of a first layer (e.g., prior toS220, prior to S230, prior to S240, etc.). In a specific example, S210includes the addition of aminosilane groups to surface (e.g., raw glasssurface) of a set of buoyant particles through incubating the set ofbuoyant particles with a solvent, wherein the set of buoyant particlesare dried prior to being introduced into the system (e.g., system 100).

4.2 Method: Adding a Set of Inputs to a Reaction Vessel S220

The method 200 can optionally include adding a set of inputs to areaction vessel S220, which functions to initiate one or more futureprocesses of the method 200.

The set of inputs preferably includes the set of buoyant particles andany other inputs required for processing, maintaining, and/or washingthe set of buoyant particles, such as any or all of: buffers (e.g.,storage buffer, wash buffer, etc.), processing materials (e.g., 1^(st)layer materials, 2^(nd) layer materials, 3^(rd) layer materials, etc.),other fluids and/or solutions (e.g., required to enable a reactionbetween the set of buoyant particles and the processing materials,water, solvents, reagents, etc.), and/or any other suitable solutionsand materials.

The set of inputs can be added to the reaction vessel through and/orfrom any or all of: a user (e.g., by a user, by a user pipetting a setof inputs into the reaction vessel, by a user pouring a set of inputsinto the reaction vessel, etc.), a component of the system 100 (e.g., acontainer via a pump and a fluidic pathway, a fluidic pathway, a filter,etc.), and/or any other suitable source. The set of inputs can be addedto the reaction vessel manually, automatically, or any combination ofboth. The set of inputs can be added to the reaction vessel separately(e.g., at multiple times, one at a time, 2^(nd) layer materials addedafter set of buoyant particles re-enters the reaction vessel from afilter, 3^(rd) layer materials added after set of buoyant particlesre-enters the filter a second time, etc.), simultaneously, or anycombination of both.

Adding a set of processing materials to the reaction vessel can includeadding materials corresponding to one or more layers (e.g., first layer,second layer, third layer, etc.) to the reaction vessel. Preferably,materials are added a single layer at a time (e.g., first layerprocessing materials added at a first time, second layer processingmaterials added at a second time after the 1^(st) layer has been formed,etc.), which can function to promote an ordered, sequential layering tothe set of buoyant particles, which can in turn function to preventnonspecific binding. The inputs are preferably added to the reactionvessel through one or more inlet ports, but can additionally oralternatively be added in any suitable way.

S220 can optionally be followed by a waiting time, which functions toenable the processing materials to completely and evenly coat thebuoyant particles. The waiting time preferably occurs while the contentsof the reaction vessel are being stirred but can additionally oralternatively be performed in the absence of stirring, in the presenceof heating, in the presence or cooling, and/or in any other environment.In some variations, for instance, the processing of the buoyantparticles (e.g., addition of a single layer) is performed for 1 hour(e.g., with stirring).

S220 can be performed a single time (e.g., just 2^(nd) layer, just3^(rd) layer, etc.) or multiple times (e.g., 2^(nd) layer followed by3^(rd) layer, 1^(st) layer followed by 2^(nd) layer, 1^(st) layerfollowed by 2^(nd) layer followed by 3^(rd) layer, etc.) throughout themethod 200.

In one variation, S220 includes adding a set of buoyant particles with afirst set of processing materials (e.g., and any accompanying solutions)to the reaction vessel 110 at a first time; adding the filtered set ofbuoyant particles having a 1^(st) layer (e.g., via the filter) and asecond set of processing materials (e.g., manually by a user) to thereaction vessel at a second time; and adding the filtered set of buoyantparticles having a 1^(st) layer and a 2^(nd) layer, and a third set ofprocessing materials to the reaction vessel at a third time.

4.3 Method: Stirring the Contents of the Reaction Vessel S230

The method includes stirring the contents (e.g., inputs) of the reactionvessel S230 (e.g., with a stirring mechanism 120), which functions tofully suspend the set of buoyant particles and promote a uniform coatingof the buoyant particles with the processing materials. S230 ispreferably performed throughout (e.g., continuously) subsequentprocesses the method 200, but can additionally or alternatively beperformed throughout the entire method 200 (e.g., during preprocessing),intermittently (e.g., at predetermined times, at random times, upondetecting a non-zero volume within the reaction vessel, etc.), or at anysuitable time(s).

S230 is preferably performed in accordance with a set of stir parameters(e.g., rotational velocity, rotational acceleration, etc.) configured tomaintain and/or enable any or all of: a constant motion of each of theset of buoyant particles, a spacing between buoyant particles above apredetermined threshold (e.g., greater than 0.1 microns, greater than0.5 microns, greater than 5 microns, greater than 10 microns, etc.), adistribution of buoyant particles (e.g., even distribution, somewhatuneven distribution, presence of buoyant particles below a surface ofthe volume in the reaction vessel, presence of buoyant particles below amiddle height of the volume in the reaction vessel, etc.), an evencoating of each of the set of buoyant particles (e.g., between 75% and100% of buoyant particles are properly coated, greater than 50% ofbuoyant particles are properly coated, etc.), and/or be otherwiseconfigured. One or more stir parameters can additionally oralternatively be determined based on any or all of: the particular setof processing materials in the reaction vessel, a time (e.g., a timerequired for buoyant particles to circulate through system, etc.), apump parameter, a height of the reaction vessel, and/or any othersuitable parameters.

In one variation, the contents of the reaction vessel are constantlystirred throughout the method by a stirring mechanism, wherein thestirring mechanism rotates at a speed between 400 and 500 rpm.Additionally or alternatively, the stirring mechanism can rotate at aspeed less than 400 rpm, greater than 500 rpm, and/or any other suitablerotational speed.

4.4 Method: Washing the Set of Buoyant Particles S240

The method 200 can include washing the set of buoyant particles S240,which functions to prepare the set of buoyant particles for additionalprocessing (e.g., addition of subsequent layer, subsequent processing,subsequent testing, etc.).

S240 is preferably performed after S230 (e.g., after a waiting time ofS230), further preferably after each iteration of S230, but canadditionally or alternatively be performed after S250, prior to S230,and/or at any other suitable time(s) during the method 200.

S240 is preferably performed in accordance with a pump (e.g., secondpump 134), wherein the pump functions to transfer a wash buffer from awash buffer container to a cavity of the reaction vessel, but canadditionally or alternatively be performed manually, with anothercomponent (e.g., of the system 100), in absence of a pump, or otherwiseperformed.

The wash buffer preferably includes phosphate and sodium chloride, butcan additionally or alternatively include any suitable solvents,reagents, solutions, detergents, or other suitable components.

In one variation, S240 includes washing the set of buoyant particlesafter each of a set of layers has been added to the buoyant particles(e.g., and prior to entering the filter).

4.5 Method: Filtering the Contents of the Reaction Vessel S250

The method includes filtering the contents of the reaction vessel S250,which functions to separate the set of buoyant particles from theremaining contents of the reaction vessel (e.g., wash buffer, processingmaterials, etc.). The remaining contents can include, for instance:remaining (e.g., unattached, extra, etc.) processing materials, such asany or all of: remaining 1^(st) layer elements, remaining 2^(nd) layerelements (e.g., linker), remaining 3^(rd) layer elements, any othersuitable layer elements, wash buffer, other buffer, a solvent, debris(e.g., fragments from broken buoyant particles), and/or any othermaterials. S250 is preferably performed multiple times throughout themethod (e.g., after each of a set of processing processes), but canalternatively be performed a single time. S250 can additionally includecollecting waste from an outlet (e.g., permeate outlet), such as in awaste chamber arranged above each of a set of filter elements.

In one variation, S250 includes passing the contents of the reactionvessel through a tangential flow filter (e.g., after S240, prior toS240, in absence of S240, etc.). In a specific example, the contents ofthe reaction vessel are transferred to the filter via a first pump 132.

4.6 Method: Repeating Any or All of the Previous Processes

The method can include repeating any or all of the above processes. Inone variation, the method 200 includes repeating S220-250 for each of aset of layers and/or other surface modifications to be added to the setof buoyant particles.

4.7 Method: Variations

In one variation, the method 200 includes: preprocessing a set ofbuoyant particles (e.g., adding an aminosilane layer to the set ofbuoyant particles and letting the buoyant particles dry); adding thepre-processed set of buoyant particles along with processing materialsassociated with a 1^(st) layer to the reaction vessel; stirring thecontents of the reaction vessel for a predetermined waiting period;washing the set of buoyant particles; filtering the set of buoyantparticles from the remaining contents of the reaction vessel;transferring the set of buoyant particles having a first layer back tothe reaction vessel; adding a set of processing materials associatedwith a 2^(nd) layer to the reaction vessel; stirring the contents of thereaction vessel for a second predetermined waiting period (e.g., thesame as the first waiting period, different than the first waitingperiod, etc.); washing the set of buoyant particles; filtering the setof buoyant particles from the remaining contents of the reaction vessel;transferring the set of buoyant particles having a first layer and asecond layer back to the reaction vessel; adding a set of processingmaterials associated with a 3^(rd) layer to the reaction vessel;stirring the contents of the reaction vessel for a predetermined waitingperiod (e.g., the same as the first waiting period, different than thefirst waiting period, the same as the second waiting period, differentthan the second waiting period,etc.); washing the set of buoyantparticles; filtering the set of buoyant particles from the remainingcontents of the reaction vessel; and collecting the set of buoyantparticles having a 1^(st), 2^(nd), and 3^(rd) layer (e.g., at acontainer).

Additionally or alternatively, the method 200 can include any othersuitable processes performed in any suitable order.

The FIGURES illustrate the architecture, functionality and operation ofpossible implementations of systems, methods and computer programproducts according to preferred embodiments, example configurations, andvariations thereof. In this regard, each block in the flowchart or blockdiagrams may represent a module, segment, step, or portion of code,which comprises one or more executable instructions for implementing thespecified logical function(s). It should also be noted that, in somealternative implementations, the functions noted in the block can occurout of the order noted in the FIGURES. For example, two blocks shown insuccession may, in fact, be executed substantially concurrently, or theblocks may sometimes be executed in the reverse order, depending uponthe functionality involved. It will also be noted that each block of theblock diagrams and/or flowchart illustration, and combinations of blocksin the block diagrams and/or flowchart illustration, can be implementedby special purpose hardware-based systems that perform the specifiedfunctions or acts, or combinations of special purpose hardware andcomputer instructions.

The method 100 and/or system 200 of the preferred embodiment can beembodied and/or implemented at least in part as machine configured toreceive a computer-readable medium storing computer-readableinstructions. The instructions are preferably executed bycomputer-executable components preferably integrated with the system andone or more portions of the processor and/or analysis engine. Thecomputer-readable medium can be stored in the cloud and/or on anysuitable computer-readable media such as RAMs, ROMs, flash memory,EEPROMs, optical devices (CD or DVD), hard drives, floppy drives, or anysuitable device. The computer-executable component is preferably ageneral or application specific processor, but any suitable dedicatedhardware or hardware/firmware combination device can alternatively oradditionally execute the instructions.

Although omitted for conciseness, the preferred embodiments includeevery combination and permutation of the various system and/or methodcomponents.

As a person skilled in the art will recognize from the previous detaileddescription and from the figures and claims, modifications and changescan be made to the preferred embodiments of the invention withoutdeparting from the scope of this invention defined in the followingclaims.

We claim:
 1. A system for processing a set of buoyant particles, thesystem comprising: a reaction vessel, the reaction vessel comprising: afirst inlet configured to receive a wash buffer; a second inletconfigured to receive a set of processing materials; a third inletconfigured to receive the set of buoyant particles; and a first outlet;a stirring subsystem comprising a stir rod and an impeller, the impellerarranged within the reaction vessel; a tangential flow filtercomprising: a set of hollow fibers, each of the set of hollow fibershaving an inner diameter larger than a diameter of the set of buoyantparticles; a fourth inlet configured to receive the set of buoyantparticles; a second outlet; a pump arranged downstream of the firstoutlet, wherein the pump comprises a diaphragm pump; and a set offluidic pathways configured to fluidly connect: the first inlet with awash buffer container, the wash buffer container comprising the washbuffer; the second inlet with a processing material container, theprocessing material container comprising the set of processingmaterials; the third inlet with the second outlet; and the first outletwith the fourth inlet.
 2. The system of claim 1, wherein each of the setof buoyant particles has a particle diameter between 10 and 30 microns,and wherein the inner diameter of each of the set of hollow fibers isgreater than the particle diameter.
 3. The system of claim 2, whereineach of the set of buoyant particles comprises glass.
 4. The system ofclaim 3, wherein each of the set of buoyant particles comprises a silicaglass coated in an aminosilane layer.
 5. The system of claim 1, whereina central axis of the fourth inlet is arranged parallel with a centralaxis of the set of hollow fibers.
 6. The system of claim 1, wherein thediaphragm pump comprises four pistons.
 7. The system of claim 1, furthercomprising a second pump, wherein the second pump comprises aperistaltic pump arranged upstream of the first inlet.
 8. The system ofclaim 1, wherein the set of processing materials comprises a firstsubset of processing materials, a second subset of processing materials,and a third subset of processing materials, wherein: the first subset ofprocessing materials forms a first layer arranged on the set of buoyantparticles; the second subset of processing materials forms a secondlayer arranged on the first layer; and the third subset of processingmaterials forms a third layer arranged on the second layer.
 9. Thesystem of claim 8, wherein: the first layer comprises a hydroxyl group;and the second layer comprises a chain of repeating glycol units. 10.The system of claim 9, wherein: the first layer comprises3-aminopropyltrimethoxysilane and an amino group; the second layercomprises an amino-reactive group, a polyethylene glycol chain, and athiol-reactive group; and the third layer comprises a thiol group.
 11. Amethod for processing a set of buoyant particles, the method comprising:a) at a reaction vessel comprising a first inlet, a second inlet, athird inlet, and a first outlet, receiving: a set of processingmaterials at the second inlet; and the set of buoyant particles at thethird inlet; b) stirring the contents of the reaction vessel with astirring subsystem comprising a stir rod and an impeller, the impellerarranged within the reaction vessel; c) receiving a wash buffer at thefirst inlet of the reaction vessel; d) pumping the contents of thereaction vessel to a tangential flow filter with a pump, wherein thepump comprises a diaphragm pump; e) at the tangential flow filtercomprising a fourth inlet, a second outlet, and set of hollow fibers,each of the set of hollow fibers having an inner diameter larger than adiameter of the set of buoyant particles: receiving the contents of thereaction vessel from the first outlet at the fourth inlet; separatingthe set of buoyant particles from a remainder of the contents of thereaction vessel; f) repeating (a) through (e) for a second set ofprocessing materials; and g) repeating (a) through (e) for a third setof processing materials.
 12. The method of claim 11, wherein each of theset of buoyant particles has a particle diameter less than 30 microns,and wherein the inner diameter of each of the set of hollow fibers isgreater than the particle diameter.
 13. The method of claim 12, whereineach of the set of buoyant particles comprises glass.
 14. The method ofclaim 13, wherein each of the set of buoyant particles comprises asilica glass coated in an aminosilane layer.
 15. The method of claim 11,wherein a central axis of the fourth inlet is arranged parallel with acentral axis of the set of hollow fibers.
 16. The method of claim 11,wherein the diaphragm pump comprises four pistons.
 17. The method ofclaim 11, wherein receiving the wash buffer at the first inlet of thereaction vessel comprises pumping the wash buffer from a wash buffercontainer to the first inlet with a second pump, wherein the second pumpcomprises a peristaltic pump arranged upstream of the first inlet. 18.The method of claim 11, wherein: the first set of processing materialsforms a first layer arranged on the set of buoyant particles; the secondset of processing materials forms a second layer arranged on the firstlayer; and the third set of processing materials forms a third layerarranged on the second layer.
 19. The method of claim 18, wherein: thefirst layer comprises a hydroxyl group; and the second layer comprises achain of repeating glycol units.
 20. The method of claim 19, wherein:the first layer comprises 3-aminopropyltrimethoxysilane and an aminogroup; the second layer comprises an amino-reactive group, apolyethylene glycol chain, and a thiol-reactive group; and the thirdlayer comprises a thiol group.
 21. The system of claim 1, wherein thepump is arranged upstream of the fourth inlet.